A point-of-care single nucleotide variation assay based on strand-displacement-triggered recombinase polymerase amplification

Abstract

The single nucleotide variation (SNV) assay holds significant value in the diagnosis of genetic diseases. However, the wide application of SNV analysis in clinics meets many difficulties because of the long test time and the limited experimental conditions. In this study, we develop a lateral flow-based strand-displacement-triggered recombinase polymerase amplification (SD-triggered RPA) assay for detecting β-thalassemia-related SNV (codon 17 (A>T) mutation), which is sensitive, fast, simple and easy to interpret in clinics. By innovatively integrating toehold-mediated strand displacement into recombinase polymerase amplification (RPA), selective strand displacement starts with the specific discrimination of SNV and triggers subsequent RPA reaction, releasing the displaced strands to contribute more D-loop structures for the strand-displacement probes to hybridize with the template strands in next amplification cycle. The limitations of current RPA SNV assays regarding specificity and sensitivity have been overcome in this method, as low as 4 pg/μL codon 17 (A>T) mutant genomic DNA (nearly 6 copies) can be accurately determined and robust behavior in clinical specimen analysis was also demonstrated. Moreover, the clinical samples can be analyzed within 35 min from sample extraction to obtaining results. Thus, the SD-triggered RPA strategy provides a pragmatic visualization approach for the rapid, sensitive, and easy detection of codon 17 (A>T) mutation, which is a promising method for point-of-care (POC) SNV assay and exhibits significant potential for future application in clinics.